Although various aspects of transfer RNA (tRNA) recognition by aminoacryl-tRNA synthetases have been well established, that of structural recognition is not understood. The study of recognition of E. coli tRNACys by cysteine-tRNA synthetase offers the potential that questions of tRNA structural recognition can be addressed. One objective is to determine the mechanism of contribution of a single nucleotide U73 to aminoacylation. A second objective is to investigate the scope and impact of a structural recognition of an unusual G15:G48 base pair that is unique to E. coli tRNACys. A third objective is to study the potentially novel mechanism of aminoacylation with cysteine in an archaebacterium. In aim 1, the hypothesis that U73 uses its functional groups to contribute to aminoacylation will be tested. In aim 2, the hypothesis that U73 uses its ability to confer a fold-back structure of the CCA end to contribute to aminoacylation will be tested. In aim 3, the biologically relevant structure of the important G15:G48 in E. coli tRNACys will be defined and tested, In aim 4, the hypothesis that G15:G48 coordinates with multiple nucleotides to contribute to aminoacylation will be tested. In aim 5, the hypothesis that tRNA structural recognition is the primary difference between the E. coli and human systems of aminoacylation with cysteine will be tested. In aim 6, the hypothesis that aminoacylation of tRNACys in the archaebacterium Halobacter volcanii (H. volcanni) is catalyzed by a novel cysteine-tRNA synthetase will be tested. The results of these studies will be integrated into structural studies of tRNACys and its complex with cysteine-tRNA synthetase in order to have a better understanding of tRNA structural recognition by an aminoacyl-tRNA synthetase.